1. Field of the Invention
The invention relates to the fields of molecular biology, bacteriology and industrial fermentation. More specifically, the invention relates to the identification and isolation of nucleic acid sequences and proteins for subunits of a novel, membrane bound sorbitol dehydrogenase in Gluconobacter suboxydans. The invention further relates to the fermentative production of L-sorbose from D-sorbitol and the subsequent production of 2-keto-L-gulonic acid.
2. Related Art
Sorbitol dehydrogenase (SDH) is an enzyme responsible for the efficient conversion of D-sorbitol into L-sorbose during sorbose fermentation in the process of the manufacturing of 2-keto-L-gulonic acid (2-KLG), an important precursor for vitamin C synthesis. Gluconobacter possesses several SDHs, which may be categorized according to their cofactor requirement: (1) NAD-dependent, (2) NADP-dependent and (3) NAD(P)-independent types. Among them, NAD(P)-independent enzyme is believed to be directly involved in efficient production of sorbose during industrial sorbose fermentation (Cummins. J. T. et al., J Biol. Chem., 224, 323; 226, 3 01 (1957)).
The process of manufacturing L-sorbose from D-sorbitol is typically performed by fermentation with an acetic acid bacterium such as Gluconobacter suboxydans and Acetobacter xylinum. At room temperature, 96-99% of conversion is made in less than 24 hours (Liebster, J. et al., Chem. List., 50:395 (1956)).
L-sorbose produced by the action of SDH is a substrate in the production of 2-keto-L-gulonic acid (2-KLG). A variety of processes for the production of 2KLG are known. For example, the fermentative production of 2-KLG via oxidation of L-sorbose to 2-KLG via a sorbosone intermediate is described for processes utilizing a wide range of bacteria: Gluconobacter oxydans (U.S. Pat. Nos. 4,935,359; 4,960,695; 5,312,741; and 5,541,108); Pseudogluconobacter saccharoketogenes (U.S. Pat. No. 4,877,735; European Pat. No. 221 707); Pseudomonas sorbosoxidans (U.S. Pat. Nos. 4,933,289 and 4,892,823); mixtures of microorganisms from these and other genera, such as Acetobacter, Bacillus, Serratia, Mycobacterium, and Streptomyces (U.S. Pat. Nos. 3,912,592; 3,907,639; and 3,234,105); and novel bacterial strains (U.S. Pat. No. 5,834,231).
A number of enzymes involved in the fermentative oxidation of sorbitol, sorbose and sorbosone are identified in the literature. U.S. Pat. Nos. 5,888,786; 5,861,292; 5,834,263 and 5,753,481 disclose nucleic acid molecules encoding and/or isolated proteins for L-sorbose dehydrogenase and L-sorbosone dehydrogenase; and U.S. Pat. No. 5,747,301 discloses an enzyme with specificity for sorbitol dehydrogenase.
In addition to distinguishing Gluconobacter SDH's on the basis of cofactor requirements, other physical characteristics may be found in the literature that distinguish these different enzymes. For example, the sorbitol dehydrogenase identified in U.S. Pat. No. 5,747,301 is distinguished on the basis of subcellular location (membrane-bound) and a haloenzyme molecular weight of 800.+-.50 kDa (10 homologous subunits of 79.+-.5 kDa). The membrane-bound D-sorbitol dehydrogenase isolated by Shinagawa et al.(E. Shinagawa, K. Matsushita, 0. Adachi and M. Ameyama (Agric. Biol. Chem., 46, 135-141, 1982)) consisted of three kinds of subunits with molecular weights of 63,000, 51,000 and 17,000.
In an effort to improve the productivity of commercial fermentation in the production of 2 KLG, the inventors have identified a novel, membrane-bound sorbital dehydrogenase in a strain of G. suboxydans that is distinct from others described in the literature (Choi, E. S. et al., FEMS Microbiol. Lett., 125:45 (1995)).